The Research Of Gene-Targeting At Gef β-Casein Gene Locus With Human IL-11 Gene

To generate IL-11 high-expression transgenic livestock, many researches were carried out on embryonic fibroblast gene-targeting assay. 1. A goat cosmid genomic library was constructed for the screening of β -casein gene. 2. The gene-targeting vector pNBCTil-11 was constructed with the cloning of IL-11 minigene downstream of β -casein promoter. 3. The C127 cell line deriving from mouse breast cancer was transfected with pNBCIL-11, and the medium was analyzed by ELISA after the G418-resistant clones were induced by prolactin, hydrocortisone and insulin. 4. After transfection of GEFs with pNBCTil-11, the cells were incubated in G418 and GANG containing medium and further analyzed by PCR and Southern blot for targeting events.3.62 X 105 recombinants were collected with an average insert size of 40kb ranging from 34kb to 45kb hi the genomic library, and the proportion of positive clones was about 97.5%. The gene-targeting vector pNBCTil-11 indicated to be expected, proved by assays of restriction enzyme digestion, sequencing, and transfection of C127 cells followed by ELISA. In the 136 clones separated, only 2 clones were tested positive by nest PCR and Southern blot.This is the first report in China regarding the construction of goat genomic library. As we know the library is the basis of the research in goat molecular genetics and gene engineering. The gene-targeting technique in primary fibroblast was successfully used in GEFs in this experiment, and the cell lines were finally obtained that the human IL-11 gene was integrated in 3 -casein gene locus of goat.